Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

Abstract

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5'-3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.

The nucleotide sequence of the gene encoding the K88ab protein subunit of procine enterotoxigenic Escherichia coli

“…Chloramphenicol was added during the early exponential phase to a final concentration of 170/~g/ml [16]. Purified plasmid DNA was obtained by CsC1 -ethidium bromide gradient centrifugation of cleared lysates prepared with Brij-58 and desoxycholate [17].…”

The nucleotide sequence of the K88ad protein subunit of porcine enterotoxigenic Escherichia coli

“…For amplification of the plasmid, chloramphenicol was added during the early exponential phase, to a final concentration of 170 /~g/ml. Purified plasmid DNA was obtained by CsC1 ethodium bromide gradient centrifugation of cleared lysates prepared with Brij58 and deoxycholate [13].…”

A gene and its product required for transposition of resistance transposon Tn2603

“…14C-amino acids (45 mCi/mmol; Commissariat a L'Energie Atomique, Paris, France), and lysis of minicells were carried out by the methods of Veltkamp et al (16). Minicell lysates were loaded on sodium dodecyl sulfate-polyacrylamide gels as described by Laemmli (11).…”